We investigate the molecular mechanisms that control the sorting of transmembrane proteins in the endosomal-lysosomal system. Sorting is mediated by interactions between signals in the cytosolic domains of the transmembrane proteins and adaptor protein associated with the cytosolic face of membranes. Two major types of sorting signals, referred to as tyrosine-based and dileucine-based, have been previously described. Work in this section has demonstrated that both types of signals are recognized with characteristic fine specificities by the adaptor protein (AP) complexes AP-1, AP-2, AP-3 and AP-4, or by the GGA adaptor proteins GGA1, GGA2 and GGA3. Mutations in AP-3 are the cause of the pigmentation and bleeding disorder, Hermansky-Pudlak syndrome type 2. Current work is aimed at elucidating the structure, regulation and physiological roles of the AP complexes and GGAs, and investigating the possibility that defects in these proteins underlie protein trafficking disorders. Over the past year, we have continued our studies on the structure and function of the GGA proteins and AP complexes. The GGAs function as Arf-dependent adaptors for the recruitment of clathrin to the TGN. In addition, they participate in the sorting of mannose 6-phosphate receptors (MPRs) and their cargo, the lysosomal hydrolases, from the TGN. Finally, they also function in the sorting of ubiqutinated transmembrane proteins at the TGN and endosomes. In collaboration with James Hurley we have recently elucidated the structural mechanism for the recognition of ubiquitin by the GAT domain of the GGAs. We have also shown that the association of one of these proteins, GGA3, with membranes is regulated by serine-phosphorylation downstream of the EGF receptor. In addition, we have performed studies on the function of AP complexes. In particular, we have shown that the AP-2 complex is critical for the delivery of lysosomal membrane proteins to lysosomes, indicating that much of this transport occurs via the plasma membrane. The characterization of the molecular machinery involved in protein sorting is important for the understanding of the pathogenesis of various metabolic and developmental disorders. An example of such a disorder is the Hermansky-Pudlak syndrome (HPS), a genetically heterogeneous heritable disease that affects lysosome-related organelles such as melanosomes and platelet dense bodies. We have previously demonstrated that mutations in the gene encoding the beta3A subunit of AP-3 are the cause of HPS type 2. In collaboration with Graca Raposo and Michael Marks we have now shown that the AP-3 complex is involved in the sorting of tyrosinase from endosomes to melanosomes, and that defects in this transport underlie HPS type 2.